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Abstract

Background and aim

Because low serum zinc levels precipitate hepatic encephalopathy, zinc supplementation
is considered a potential therapeutic option. The aim of this study was to assess
the effects of oral zinc supplementation in the treatment of hepatic encephalopathy.

Methods

For this systematic review and meta-analysis, data sources included electronic databases
(CENTRAL, MEDLINE, EMBASE) and manual searching. Randomized clinical trials of adult
patients diagnosed with liver cirrhosis and hepatic encephalopathy were included.
The types of interventions considered were any oral zinc supplementation versus no
intervention, placebo, or other interventions for the management of hepatic encephalopathy.
The data were analyzed by calculating the RR for each trial and expressing the uncertainty
as 95% CI. Continuous data were analyzed by calculating the standard mean differences
(SMD) between groups within each trial and their 95% CI. Statistical heterogeneity
was defined as a P-value > 0.10 (χ2) or I2 > 25%.

Results

Four trials with a total of 233 patients were included. Oral zinc supplementation
was associated with a significant improvement in performance on the number connection
test (SMD –0.62; 95% CI –1.12 to –0.11) reported in three trials (n = 189), but not
with encephalopathy recurrence reduction (RR 0.64; 95% CI 0.26 to 1.59) reported in
two trials (n = 169). Other clinically significant outcomes (mortality, liver related
morbidity, quality of life) were not reported.

Conclusion

Oral zinc supplementation improved performance on the number connection test, but
no evidence about other clinical or biochemical outcomes was available.

Keywords:

Therapy; Liver cirrhosis; Evidence-based medicine

Introduction

Hepatic encephalopathy is a neuropsychiatric complication of liver disease that affects
20 to 30% of the patients with cirrhosis [1,2], reducing health-related quality of life and causing a reversible decline in cognitive
function. Previous studies have demonstrated that a reduction in blood ammonia levels
improves hepatic encephalopathy, neuropsychological test performance, cognitive function,
and health-related quality of life [3]. Lactulose, an ammonia absorption minimizer, has been successfully used to reduce
blood ammonia levels in minimal hepatic encephalopathy. However, lactulose has no
ammonia detoxification effect, rendering it ineffective to treat advanced hepatic
encephalopathy [4-6].

Two major organs are involved in the metabolism of ammonia: the liver, in which ammonia
is converted to urea via ornithine transcarbamylase, and the skeletal muscle, where
ammonia is metabolized to glutamic acid via glutamine synthetase [5]. Zinc is a critical cofactor in these enzymatic reactions. Animal models have shown
zinc deficiency decreases the activity of ornithine transcarbamylase, while zinc supplementation
markedly increases hepatic ornithine transcarbamylase activity. Zinc deficiency has
also been reported to impair the activity of muscle glutamine synthetase, which leads
to hyperammonemia [6-8].

Zinc deficiency is observed frequently in patients with cirrhosis and hepatic encephalopathy
[9]. Poor nutritional intake caused by a protein-restricted diet, impaired intestinal
absorption, and excessive urinary loss are all potential causes of a low serum zinc
levels in patients with advanced cirrhosis [5]. Short-term oral zinc supplementation may improve hepatic encephalopathy by correcting
the zinc deficiency that compromises the conversion of ammonia to urea [10]. Bresci et al. reported better psychometric test performance in a zinc-supplemented
group than in a standard therapy group, although the difference was not significant
[11]. Similarly, oral zinc supplementation can improve hepatic encephalopathy in patients
failing to respond to protein restriction and lactulose [2,6-8].

Zinc supplementation, in addition to standard therapies, may increase the hepatic
conversion of amino acids into urea, decrease serum ammonia level, and consequently
improve health-related quality of life. The effect of long-term oral zinc supplementation
in addition to standard therapy on recurrent hepatic encephalopathy has not been established
[7,8,12]. Despite the low cost and infrequent side effects of zinc supplementation, there
is little evidence-based information about the effects of zinc supplementation on
hepatic encephalopathy. The aim of this meta-analysis was to assess the effects of
oral zinc supplementation in the treatment of hepatic encephalopathy.

Methods

Types of studies

Prospective randomized clinical trials that compared the effects of zinc supplementation
with those of no intervention, placebo, or standard therapy on hepatic encephalopathy
in patients with liver cirrhosis were included. Trials were included irrespective
of publication status, year of publication, or language.

Types of participants

All adult patients diagnosed with liver cirrhosis using a combination of biochemical
and clinical data, regardless of the etiology and treatment, diagnosed with hyperammonemia
and hepatic encephalopathy were included.

Types of interventions

Studies that compared oral zinc supplementation with no intervention, placebo, or
other interventions for the management of hepatic encephalopathy were included.

Types of outcome measures

The primary outcome measures were all-cause mortality, disease-specific mortality
(mortality secondary to complications of liver cirrhosis), and severity of encephalopathy
as assessed by performance on neuropsychometric tests or recurrence.

The secondary outcome measures were adverse events (all types of adverse events) and
quality of life score (measured by any scale).

Search methods for identification of studies

Electronic searches

Relevant randomized trials were identified by searching in CENTRAL, MEDLINE, and EMBASE.

Searching other resources

The references in all identified studies were inspected to identify other trials.
The first or corresponding author of each included trial, as well as active researchers
in the field were contacted for information about unpublished trials and additional
information from their own trials.

Selection of studies

Two authors independently inspected each identified reference and applied the inclusion
criteria. For potentially relevant articles or in cases of disagreement between the
two reviewers, the full-text article was obtained and inspected independently; if
the disagreement could not be solved, a third author reviewed the article. Justification
for study exclusion was documented.

Data extraction and management

Two authors independently extracted the data from the included trials. In cases of
disagreement, a third author extracted the data. Extracted data were discussed and
this discussion was documented; when necessary, the authors of the original studies
were contacted. Justification for study exclusion was documented. Trials were identified
by the last name of the first author and the year of publication.

Assessment of risk of bias in included studies

Two authors independently assessed risk of bias in the trials without masking the
trial names. Assessment was conducted according to the Cochrane Handbook for Systematic
Reviews of Interventions [13].

Measures of treatment effect and data analysis

RevMan Analyses software was used for the statistical analysis [14]. Dichotomous data were synthesized and analyzed by calculating the RR and 95% CI
for each trial. Continuous data were synthesized and analyzed by calculating the standard
mean difference (SMD) between groups for each trial and its 95% CI.

Assessment of heterogeneity

We checked the heterogeneity of effects across trials by visual inspection of the
forest plots and χ2 and I2 tests for heterogeneity. Statistical heterogeneity was defined as P > 0.10 (χ2) or I2 > 25%. Whenever heterogeneity was detected, subgroup analysis was performed to assess
the effect of potential sources of heterogeneity on the main results.

Assessment of reporting biases

A funnel plot estimating the precision of trials (plot of logarithm of the RR against
the sample size) was used to evaluate asymmetry and detect potential publication bias.
In addition, Egger´s test was used to quantify the bias captured by the funnel plot
[15].

Sensitivity analysis

We analyzed the data using both fixed and random-effect models. When both models produced
similar estimates, the fixed-effect result was reported; otherwise, we reported the
results from both analyses (Additional file 1: Figure S1). Outcomes were analyzed as reported in the trial, either per protocol
or as an intention-to-treat.

Results

Study selection

A total of 65 potential references were retrieved: 36 were narrative reviews, 15 were
nonrandomized studies, four were symposium reviews, one was a systematic review of
different target trials, one was a clinical trial in animals, one was a trial in children,
one was a clinical guideline, and one was a book chapter. Finally, five randomized
controlled trials were included in the first analysis, but one study was excluded
after a second evaluation of the inclusion criteria (Figure 1).

Study characteristics

We included four randomized controlled trials designed to evaluate oral zinc supplementation
in the treatment of hepatic encephalopathy. The number of patients who received oral
zinc supplementation ranged from 20 to 90. A total of 233 patients from three countries,
Belgium [10], Italy [11], and Japan [1,16], were included. All studies involved patients with cirrhosis and different stages
of encephalopathy. The doses used were zinc sulfate 600 mg/d [16], zinc acetate 600 mg/d [10,11], or polaprezinc 225 mg/d [1] (containing 51 mg of zinc and 174 mg of L-carnosine). All studies were randomized, double-blind, placebo-controlled trials
(Table 1).

Table 1.Characteristics of trials included in this systematic review and meta-analysis

Risk of bias within studies

The risk of bias was unclear in all trials. Lack of information precluded a proper
evaluation of the risk of bias for all studies.

Synthesis of results

Given the large heterogeneity of outcomes across studies, the meta-analysis was restricted
to two primary outcomes: number connection test performance and rate of encephalopathy
recurrence. Patients treated with oral zinc supplementation experienced a significant
improvement in the number connection test performance (SMD –0.62; 95% CI –1.12 to
–0.11) compared with patients in the placebo or no supplementation groups (Figure 2). Some heterogeneity of effects (I2 = 50%) was observed, and stratified analyses were conducted by year of the study
and sample size, but no change in the direction or significance of the effect was
observed (data not shown). The funnel plot shows no evidence of publication bias (Additional
file 2: Figure S2). No reduction was observed in the encephalopathy recurrence rate (RR
0.64; 95% CI 0.26 to 1.59) (Figure 3).

Reding et al. [10] studied the use of oral zinc supplementation in a double-blind randomized trial involving
22 patients with chronic encephalopathy. The zinc group received zinc acetate 600
mg/d. Compared to placebo, the zinc group showed improved performance in the number
connection test (56 ± 25.4 and 42.12 ± 16.2 seconds, respectively).

Bresci et al. [11] assessed the effect of long-term zinc supplementation in 90 patients with cirrhosis
with stable recurrent hepatic encephalopathy. Oral zinc supplementation (zinc acetate
600 mg/d) in addition to standard therapy normalized the serum zinc levels. Performance
in the number connection test (40 ± 8 vs. 50 ±12 seconds), as well as in the portal
systemic encephalopathy index improved in the treated compared to placebo group (0.15
vs. 0.19). The treated group experienced less recurrence of encephalopathy; after
six months 88.6% of patients in the treated group had no detectable signs of hepatic
encephalopathy, compared to 86% in the placebo group.

Discussion

In this meta-analysis, we included four randomized controlled trials evaluating the
effect of oral zinc supplementation over hepatic encephalopathy. Three studies reported
data on number connection test; all three showed an improvement in performance in
the zinc group compared to placebo or standard therapy. This improvement suggests
a beneficial effect of oral zinc in encephalopathy patients. Two studies reported
data on encephalopathy recurrence rate. Both studies observed lower recurrence rates
in the zinc groups, suggesting a beneficial effect of zinc; however, given the small
sample size, confidence intervals were wide and failed to reach statistical significance.

Hepatic encephalopathy is characterized at the neurophysiological level by disturbed
corticocortical and corticomuscular coupling, and at the cellular level by primary
gliopathy [2,5,17,18]. Ammonia is a key pathophysiological factor in hepatic encephalopathy [18,19]. In the brain, ammonia is detoxified by astrocytes through a reaction catalyzed by
glutamine synthetase; an increased brain glutamine/glutamate ratio is associated with
decreased myoinositol, reflecting compensation for glial edema [20-23]. Swollen astrocytes predispose to neuronal dysfunction by impairing their regulatory
activity against the increase in protein tyrosine nitration and the formation of reactive
oxygen and nitrogen oxide species including nitric oxide. If not counteracted, these
reactions promote RNA oxidation, which prompts gene expression and the transcription
of altered proteins [2,5,6,18,19,21,24].

Cytokines or lipopolysaccharides could induce the formation of nitrogen oxide species
and trigger zinc release from metallothioneins, the principal zinc storage protein.
A fluctuation in intracellular zinc levels modulates signal transduction, transcription
factor activity, and gene expression, causing hepatic encephalopathy symptoms. Zinc
deficiency is associated with disturbances in learning, memory, and emotional stability
and is accompanied by hyperammonemia. Zinc supplementation has shown to reduce ammonia
levels in experimental animals and humans through hepatic urea synthesis stimulation
and glutamine synthesis in skeletal muscle [2,6-8,12,18,19,21,25].

The present meta-analysis is limited by the small number and poor quality of trials
included. Available trials studied heterogeneous outcomes and failed to measure critical
outcomes such as quality of life. This hinders the ability to draw conclusions about
the value of oral zinc supplementation in the treatment of hepatic encephalopathy.
Additionally, little information regarding the clinical importance of the different
zinc formulations used in the trials was available.

In conclusion, oral zinc supplementation improved performance on the number connection
test, but there is no clear evidence that supplementation improves encephalopathy
or encephalopathy-related quality of life. More trials are needed to evaluate the
use of oral zinc supplementation in patients with liver cirrhosis and hepatic encephalopathy.

Competing interests

The authors declare that no competing interests exist.

Authors’ contributions

NCC-T: protocol writing, searching, trial selection, data extraction, report writing,
drafting the article, and final approval of the manuscript. AC-A: protocol writing,
searching, trial selection, data extraction, report writing, drafting the article,
and final approval of the manuscript. TB-G: report writing, drafting the article,
and final approval of the manuscript. NM-S: report writing, drafting the article,
and final approval of the manuscript. MU\: report writing, drafting the article, and
final approval of the manuscript. All authors read and approved the final manuscript.

Financial disclosure

This manuscript was partially supported by an educational grant from Medica Sur Clinic
& Foundation. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.